Plasma samples collected on −1, 14, 20, and 26 dpi were subjected to an enzyme-linked immunosorbent assay (ELISA) developed in-house to quantify TAFV GP-specific immunoglobulin G (IgG). Briefly, 96-well half-area high binding plates (Corning, NY, USA) were coated overnight at 4 °C with 1 μg/mL TAFV GP (described above) diluted in 50 mM carbonate buffer (pH 9.6). The following day, plates were blocked for 1 h at 37 °C with 5% (w/v) skim milk (BD, Franklin Lakes, NJ, USA) diluted in phosphate-buffered saline (PBS) containing 1% (v/v) Tween-20 (1% PBST). Plasma samples were serially diluted 10-fold in 1% (w/v) bovine-serum albumin (BSA) prepared in PBS (1% BSA) starting at a dilution of 1:400 for all samples except pre-bleed (−1 dpi), for which the initial dilution was 1:100. After blocking, the plates were gently blotted, 30 μL diluted plasma was added to wells in triplicate, and plates were incubated for 1 h at 37 °C. Plates were then washed 4 times with 150 μL wash buffer (0.1% PBST) using a microplate washer. After washing, plates were blotted to remove residual wash buffer and subsequently incubated for 1 h at 37 °C with 30 μL/well HRP-conjugated goat anti-ferret IgG secondary antibody (Bethyl laboratories, Montgomery, TX, USA) diluted 1:10,000 in 1% BSA. Plates were then washed as described and incubated in the dark with 50 μL/well TMB (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. The optical density (OD) was recorded at 650 nm using the Synergy HTX plate reader (Bio-Tek, Winooski, VT, USA). The OD cut-off was defined as the average of the pre-bleeds (1/100 dilution) plus 3 standard deviations.

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